Analysis of endothelial progenitor cell subtypes as clinical biomarkers for elderly patients with ischaemic stroke

Endothelial progenitor cells (EPCs), expressing markers for stemness (CD34), immaturity (CD133) and endothelial maturity (KDR), may determine the extent of post-stroke vascular repair. Given the prevalence of stroke in elderly, this study explored whether variations in plasmatic availability of certain EPC subtypes could predict the severity and outcome of disease in older patients. Blood samples were collected from eighty-one consented patients (≥ 65 years) at admission and days 7, 30 and 90 post-stroke. EPCs were counted with flow cytometry. Stroke severity and outcome were assessed using the National Institutes of Health Stroke Scale, Barthel Index and modified Rankin Scale. The levels of key elements known to affect EPC characteristics were measured by ELISA. Diminished total antioxidant capacity and CD34 + KDR + and CD133 + KDR + counts in early phases of stroke were associated with disease severity and worse functional outcome at day 90 post-stroke. Baseline levels of angiogenic agent PDGF-BB, but not VEGF, positively correlated with CD34 + KDR + numbers at day 90. Baseline LDL-cholesterol levels were inversely correlated with CD34 + KDR+, CD133 + KDR + and CD34 + CD133 + KDR + numbers at day 90. Close correlation between baseline CD34 + KDR + and CD133 + KDR + counts and the outcome of stroke proposes these particular EPC subtypes as potential prognostic markers for ischaemic stroke.

Ischaemic stroke (IS) develops through an interference with blood supply to the brain and continues to be one of the leading causes of mortality and morbidity in the world 1 .The prevalence of stroke doubles every 10 years after the age of 55 and about two-third of all stroke patients are older than 65 years 1,2 .Although various mechanisms can initiate and exacerbate the development of atherosclerotic disease throughout chronological ageing, endothelial dysfunction (ED), accompanied by impaired vascular relaxation and inflammation, is widely considered as a key pathology behind the structural and functional alterations in the vascular system 3 .
The endothelium is important in preserving vascular homeostasis 4,5 .Endothelial progenitor cells (EPCs), released from bone marrow, play an instrumental role in maintaining appropriate endothelial function by re-endothelialisation of cerebral blood vessels after IS 6,7 .Similar to embryonic angioblasts, EPCs are equipped with an inherent capacity to proliferate, migrate and differentiate.Hence, non-haematopoietic cells (CD45-) expressing a variety of markers for stemness (CD34+), immaturity (CD133+) and endothelial cell maturity (KDR+) are regarded as distinct EPC subtypes in circulation 7,8 .Circulating EPCs can also negate the deleterious effects of cerebral ischaemia by inducing angiogenesis and vasculogenesis through secretion of various trophic factors 9 .However, the mobilisation, survival and the reparative capacity of EPCs are negatively affected by chronological ageing in that age-related changes in the availability of circulatory cytokines, chemokines and growth factors, appear to play an instrumental role 10 .Besides, advancing age renders EPCs susceptible to internal changes and environmental factors such as oxidative stress which, markedly influences the number of circulating EPCs and contributes to age-mediated gradual loss of endothelium in cerebrovasculature and to the pathogenesis of IS 11 .
Oxidative stress represents a common pathology during the acute phase of an IS and is characterised by the excessive availability of reactive oxygen species (ROS) 12 .Both the enhanced production and reduced metabolism of ROS may account for this phenomenon.Exposure to excessive levels of ROS irreversibly damages macromolecules, including DNA and proteins, exacerbates ED and may adversely affect EPC characteristics 13 .
In light of the above, we hypothesised that variations in numbers of different EPC subtypes might correlate with the severity and outcome of IS and therefore serve as prognostic markers for stroke.In addition, the study also investigated the correlation between the plasmatic profile of inflammatory cytokines, growth factors and angiogenic factors known to affect EPC characteristics and disease severity and outcome.

Study participants
Data from eighty-one older (≥ 65 years old) IS patients recruited for The Dunhill Medical Trust EPC study (DMT EPC study, NCT02980354) between February 2017 and November 2019 were used for this substudy.IS was defined as a sudden focal neurological deficit persisting longer than 24 h with no evidence of cerebral haemorrhage on imaging.The DMT EPC study was a single-centre, prospective, observational, case-controlled study, performed in accordance with the ethical standards for human experimentation established by the Declaration of Helsinki.Details of the study have already been reported elsewhere 14 .The study protocol was reviewed and approved by West Midlands-Coventry & Warwickshire Research Ethics Committee (REC number: 16/WM/0304).Written informed consent was obtained from all participants for their anonymised information to be published in this study.
National Institutes of Health Stroke Scale (NIHSS), modified Rankin Scale (mRS) and Barthel Index (BI) were used to measure patients' neurological and functional status on admission (baseline, BL) and days 7, 30 and 90 post-stroke.As all patients received guideline-based medical therapy and underwent an appropriate rehabilitation programme, patients' EPC counts and neurological outcomes were unlikely to be differently affected by these treatments.

Blood sampling and flow cytometry
Blood samples (~ 30 mL) were collected from participants at the abovementioned time points to cover acute (within the first 48 h of stroke, baseline, BL), subacute (day 7, D7) and chronic (days 30 and 90, D30 and D90, respectively) phases of stroke.Six mL of blood samples were used to count circulating EPC subtypes by flow cytometry where non-haematopoietic cells (CD45-) co-expressing two or more cell surface markers for stemness (CD34 +), immaturity (CD133 +) and endothelial maturity (KDR +), i.e., CD45-CD34 + CD133 +, CD45-CD34 + KDR +, CD45-CD133 + KDR + and CD45-CD133 + CD34 + KDR +, were counted.To determine the percentage of each EPC subpopulation 1 million total events were acquired.The researchers who carried out the experiments remained blinded to subject characteristics throughout the study to avoid bias.

Statistical analysis
The statistical analyses were performed using SPSS package version 26 (SPSS Inc., USA) and GraphPad Prism 8.0 statistical software package (GraphPad Software Inc).Spearman correlation analysis was performed to capture the association between all the variables.Spearman's correlation coefficients (r) were utilised to summarise the relationship between all the variables.p < 0.05 was considered as significant.

Ethics approval
The Dunhill Medical Trust EPC study protocol was reviewed and approved by West Midlands-Coventry & Warwickshire Research Ethics Committee (16/WM/0304).

Study population
Blood samples were collected from a total of 81 subjects with lacunar (n = 38) or cortical (n = 43) stroke on the basis of clinical syndrome and neuroimaging of whom 69% were male (n = 56).The median age at the diagnosis was 76 years.More than half of the patients had a history of smoking (n = 48), consumed alcohol (n = 51) and were hypertensive (n = 49).Over a quarter of all patients had atrial fibrillation (AF, n = 23) and were hyperlipidaemic (n = 24).Over 15% of all patients also had a previous history of transient ischaemic attack (TIA, n = 15) and IS (n = 12) and were diabetic (n = 17).Hence, over 40% of patients were on statins (n = 38), ACE inhibitors (n = 34) and anti-platelets (n = 33).The use of calcium channel blockers (n = 17), beta blockers (n = 15) and glucoselowering (n = 13) agents amongst patients was frequent (Table 1).

Analyses of correlation between biochemical parameters and stroke severity and outcome
Assessment of the plasmatic biochemical profile of IS patients showed no correlation between the BL levels of angiogenic modulators VEGF, PDGF-BB, thrombospondin-1/2, angiostatin and endostatin and the severity and outcome of stroke at D90 (Fig. 2).Similarly, the BL levels of TAC and plasmatic SDF-1 and G-CSF had no impact on any of the neurological and functional parameters.However, a negative correlation was detected by BL TNF-α levels and D90 NIHSS (r: − 0.343; p: 0.014).
Scrutiny of the correlation between circulating EPC subtypes and the levels of key elements capable of affecting their counts showed that only BL level of angiogenic factor PDGF-BB (r: 0.334; p: 0.043) increased CD34 + KDR + numbers on D90 after stroke.In contrast, no correlation between any EPC subtype and BL levels of VEGF, thrombospondin-1/2 and endostatin were observed (Fig. 3).Again, no correlation between BL levels TAC, G-CSF, SDF-1 and TNF-α and EPC counts were observed D90 post-stroke.
Analysis of correlations between D7 and D30 levels of the same biochemical elements and the severity and outcome of stroke on D90 revealed negative correlations between D7 TAC and D90 NIHSS (r: − 0.318; p: 0.028) and between D30 thrombospondin-2 and D90 mRS (r: − 0.336; p: 0.045) scores.Interestingly, D30 thrombospondin-1 levels appeared to correlate positively with the extent of disability on D90 (r: 0.459; p: 0.007) as attested by the BI score (Supplementary Table 3).The actual level of biomedical parameters at BL, D7 and D30 and the neurological and functional scores on D90 are documented in the Supplementary Tables 4 and 5, respectively.www.nature.com/scientificreports/

Discussion
Alterations in endothelial integrity and function play a critical role in the pathogenesis of stroke and develop in otherwise healthy older individuals as part of physiological ageing process 15,16 .Besides physical damage and endothelial cell senescence, diminished availability of EPCs may also contribute to age-and stroke-mediated ED and associated vascular and functional abnormalities.Substantial decreases reported in the number of circulating EPCs expressing markers specifically for stemness and immaturity in healthy individuals over 65 years of age versus those between 18 and 64 years confirm the influence of ageing on distinct EPC subtypes 10 .In light of these and further findings implying the value of circulating EPCs for endothelial repair 6,11,17 and as potential prognostic biomarkers 18 , the present study hypothesised that changes in circulating numbers of certain EPC subtypes, defined as cells co-expressing a variety of markers for stemness (CD34 +), immaturity (CD133 +) and endothelial cell maturity (KDR +), may explain the differences reported in the severity and/or outcome of  ischaemic stroke in older patients.Observation of a significant correlation in the current study between higher CD34 + KDR + and CD133 + KDR + numbers during the acute phase of stroke and better neurological and functional outcome on D90 post-stroke corroborate this hypothesis and attribute a key role to these particular EPC subtypes in neurovascular repair 19,20 .Moreover, these findings also indicate the capacity of these particular cells to serve as prognostic markers for IS patients 21 .Since one-fourth of all mild ischaemic stroke patients manifest functional changes between days 30 and 90 after stroke, it was important to assess the level of recovery and correlate the potential changes in various biochemical compounds to recovery on D90 in this study.Indeed, neurological and functional assessments performed on D7 and D30 appear to insufficiently represent long-term recovery in mild stroke 22 .The capacity of CD34 + KDR + cells to fully differentiate into mature endothelial cells has long been recognised 23 .Considering that the vasculoprotective effects of CD34 + cells may be explained by the paracrine effect rather than the ability of these cells to differentiate into fully functional endothelial cells, CD34 + cells continue to face scrutiny as markers of EPCs 24 .In contrast, mature endothelial cells do not express a great deal of CD133 antigens.The cells that co-express CD133 + and KDR + represent a phenotypically and functionally distinct subset of circulating endothelial cells and are widely regarded as true EPCs 25 .Observation of intracellular CD133 expression in EPCs and the diminished post-ischemic revascularisation in a nude mouse model with hind-limb ischemia following the administration of OECs transfected with specific siRNA (siCD133-EPCs) help reconcile the discrepancies regarding CD133 positivity and ontogeny in endothelial progenitors 26 .
Major growth factors such as VEGF 27 and PDGF-BB 28 and anti-angiogenic elements, including thrombospondin-1/2 29 , endostatin 30 and angiostatin 31 that regulate post-ischaemic vasculogenesis and angiogenesis may also determine the extent of neurological recovery.Unlike a recent study correlating BL plasma endostatin levels with increased risk of mortality and severe disability at 3 months, no correlation was observed between the BL plasma levels of these elements and NIHSS, mRS and BI scores at D90 in this study 32 .Although genetic variations between the two study populations, European vs Chinese, may somewhat account for the differences noted, the dichotomy in data cast doubt on the capacity of these agents to serve as highly specific and reliable prognostic markers for acute IS.
Environmental changes evoked by advanced age and ischaemic injury may suppress the production of EPCs and therefore exacerbate patients' dependence and disability 10,11 .Oxidative stress represents one such change and evokes cerebrovascular dysfunction by not only disrupting major cellular components but also neutralising nitric oxide, a potent anti-atherogenic agent 33,34 .Albeit insignificant, the presence of a correlation between higher levels of plasma TAC and better post-stroke neurological outcome substantiates the disruptive effects of ROS on neurovasculature and pinpoints the importance of TAC in maintaining neurovascular equilibrium.Normalisation of vascular tone in dysfunctional arterial segments by ROS scavengers and antioxidant vitamins further confirm the crucial role of oxidative stress in ED 35,36 .Under normal conditions, EPCs are better protected against oxidative injury due to possession of high TAC 37,38 , but prolonged exposure to oxidative stress adversely affect their ability to proliferate, differentiate, self-renew and secrete agents with vasculoprotective capacity such as SDF-1 and VEGF 9,12,20,39 .
In addition to production, the function of EPCs may also be adversely affected by the ageing process in that suppression of systemic elements known to affect neovasculogenesis may play a crucial role 17,40 .Indeed, observation of a positive correlation between acute plasma level of PDGF-BB and CD133 + KDR +, CD34 + KDR + numbers supports this notion.The steady increases in plasma levels of PDGF-BB are likely to trigger proliferation and directed migration of EPCs to the site of injury during chronic phases of IS to counter ischaemic damage.The absence of a correlation between early SDF-1 levels and EPC counts during chronic phases of IS may be instrumental, in this context, to keep vascular growth in check 41,42 .Although SDF-1 strongly mobilizes the release of EPCs from the bone marrow 43 , the seemingly unaltered levels of SDF-1 after IS and age-mediated potential decreases in SDF-1 receptor, CXCR4 levels may explain the abovementioned lack of correlation 18 .Overall, the timing and the degree of variations observed for the parameters studied here rule out their use as reliable diagnostic markers in clinical settings.
Considering that all study participants enrolled to the study were ≥ 65 years of age, the presence of additional vascular risk factors alongside the use of certain medicines e.g.anti-platelets and anti-hypertensives may somewhat account for the differences observed in EPC counts and patients' outcome 44,45 .Co-analyses of EPC counts with a large number of BL demographic and medical variables showed negative correlations between BL LDL-C and most EPC subtypes and specifically between BL cholesterol and CD34 + KDR + cells, confirming the point that statins significantly enhance circulating EPC numbers and pinpointing the importance of a therapy targeting cholesterol levels in stroke patients 46 .However, a recent study revealing decreases in plasma VEGF levels after statin therapy necessitate a cautious approach as regards the use of statins while signifying the importance of the type of statin used and the length of treatment received 47 .In contrast, a positive correlation between BL glucose and D90 endostatin levels indicate a prerequisite for the close monitoring of the blood glucose levels in IS patients.Positive correlations between glucose levels and endostatin levels have been established in other diseases such as diabetes and coronary artery disease 48 .
This study investigated how differences in the plasmatic availability of certain EPC subtypes during acute phase of IS might correlate with severity of disease, patients' functional outcome and the level of agents known to affect their bioavailability.Since the numbers of patients recruited for the DMT EPC study (81 patients) and retained until D90 (62 patients) were below the originally planned number of recruits (100 patients), it is possible that the reduced sample size could have resulted in lower statistical power and might have somewhat biased the results.A bigger sample size would have been useful to better compare the differences in a large number of parameters studied.Besides, given that the majority of patients enrolled for the study had significant recovery on D90 after stroke, it is important to further investigate the correlations established in this study in patients who continue to show serious morbidity on D90.
In conclusion, circulating baseline levels of CD34 + KDR + and to a lesser degree CD133 + KDR + closely associate with the outcome of IS and may be considered as prognostic markers for IS.A future study enrolling larger numbers of patients with different degrees of morbidity is required to dis/prove this point.

Figure 2 .
Figure 2. Correlation between baseline levels of angiogenic factors, total anti-oxidant capacity, chemokines and cytokines and post-stroke severity and outcome on day 90.The size of bubbles indicates the magnitude of correlation coefficient.While red and blue bubbles successively indicate significant (p < 0.05) and insignificant (p > 0.05) correlations, the symbols + and − indicate positive and negative correlations, respectively.NIHSS National Institutes of Health Stroke Scale, BI Barthel index, mRS modified Rankin Score, CD cluster differentiation, G-CSF granulocyte colony-stimulating factor, PDGF-BB platelet-derived growth factor, SDF-1 stromal cell-derived factor-1, TAC total anti-oxidant capacity, TNF-α tumour necrosis factor-α, THR-1 thrombospondin-1, THR-2 thrombospondin-2, VEGF vascular endothelial growth factor.

Figure 3 .
Figure 3. Correlation between baseline levels of angiogenic factors, total anti-oxidant capacity, chemokines and cytokines and the number of circulating EPC subtypes on day 90.The size of bubbles indicated the magnitude of correlation coefficient.While red and blue bubbles successively indicate significant (p < 0.05) and insignificant (p > 0.05) correlations, the symbols + and − indicate positive and negative correlations, respectively.CD cluster differentiation, G-CSF granulocyte colony-stimulating factor, PDGF-BB platelet-derived growth factor, SDF-1 stromal cell-derived factor-1, TAC total anti-oxidant capacity, TNF-α tumour necrosis factor-α, THR-1 thrombospondin-1, THR-2 thrombospondin-2, VEGF vascular endothelial growth factor.

Figure 4 .
Figure 4. Correlation between baseline levels of biological variables, including body mass index (BMI), blood pressure (BP) and blood triglyceride, glucose and LDL-and HDL-cholesterol and the number of circulating EPC subtypes on day 90.The size of bubbles indicated the magnitude of correlation coefficient.While red and blue bubbles successively indicate significant (p < 0.05) and insignificant (p > 0.05) correlations, the symbols + and − indicate positive and negative correlations, respectively.

Figure 5 .
Figure 5. Correlation between baseline levels of biological variables, including body mass index (BMI), blood pressure (BP) and blood triglyceride, glucose and LDL-and HDL-cholesterol and the levels of angiogenic factors, total anti-oxidant capacity, cytokines and chemokines on day 90.The size of bubbles indicated the magnitude of correlation coefficient.While red and blue bubbles successively indicate significant (p < 0.05) and insignificant (p > 0.05) correlations, the symbols + and − indicate positive and negative correlations, respectively.G-CSF granulocyte colony-stimulating factor, PDGF-BB platelet-derived growth factor, SDF-1 stromal cellderived factor-1, TAC total anti-oxidant capacity, TNF-α tumour necrosis factor-α, THR-1 thrombospondin-1, THR-2 thrombospondin-2, VEGF vascular endothelial growth factor.

Table 1 .
Demographic and baseline clinical characteristics of the patients with ischaemic stroke.BI Barthel index, CD cluster differentiation, mRS modified ranking score, NIHSS national institutes of health stroke scale.